1. Field of the Invention
This invention relates to an improved method for conducting solid phase in vitro diagnostic assays.
2. Description of the Prior Art
In recent years, numerous techniques have been employed in the area of laboratory diagnostics to simplify operating procedures of existing methods and to provide new methods of improved speed, sensitivity, and accuracy. In particular, solid phase reactions have been especially valuable in simplifying the manipulations of prior art procedures and making possible procedures that could not be performed with conventional homogeneous phase reactions.
A solid phase reaction is generally carried out between one reactant, the fixed component, immobilized on the surface of an insoluble support matrix, and a second reactant, the mobile component, in solution. The reaction occurs when a molecule or a molecular arrangement of the mobile reactant, in the course of diffusion, collides with a molecule of the fixed reactant immobilized on the surface of the solid support matrix. The reaction may be a conventional chemical reaction, a binding of the mobile component by the fixed component as in an immunochemical reaction between an antigen and an antibody, or it may be a binding of the mobile component by the fixed component accompanied by chemical transformation of one of the components such as occurs in an enzyme-catalyzed reaction. Quantitative results are obtained by measuring the formation of products or disappearance of reactants as in the case of conventional and enzyme-catalyzed reactions, and in measuring the amount of the mobile component bound or the amount of mobile component unbound, in the case of an immunochemical reaction.
Any conventional chemical reaction or enzyme-catalyzed reaction resulting in a directly or indirectly measurable change can, in principle, be carried out by solid phase techniques. Directly measurable changes include changes in pH, light absorbance in the visible and ultraviolet regions or changes in fluorescence intensity. Indirect measurements can be made whenever the primary reactants or products are not readily measurable themselves by interposing the action of a reagent to carry out further reaction steps resulting in a measurable change and by the introduction of specific separation techniques. Such strategies may be employed alone or in combination, as is understood in the art.
Where the reaction consists solely of binding, in the absence of chemical change, techniques developed in the field of immunochemistry may be used to measure the extent of the reaction. Solid phase reactions are especially suited for immunochemical assays because the reactants in bound form may readily be removed from the solution by virtue of their attachment to the solid phase. Frequently, however, the components bound in an immunochemical reaction cannot be directly measured because they are indistinguishable by chemical methods from other substances commonly present in the same reaction mixture, so that the mere disappearance of a reactive component from solution or its accumulation on the solid phase cannot be measured directly. Therefore, additional steps must be taken in order to make a measurable change related to the amount of binding.
The variety of approaches taken by workers in the prior art can be grouped into two general categories. In the first of these, termed competitive or indirect immunoassays, the immobilized component is present in controlled amount and the mobile component present in unknown amount. To the unknown amount of mobile component is added a known amount of the same component which has been tagged by the addition of a measurable substituent which does not interfere with its immunochemical reactive properties. The tag may consist of a radioisotope, a chromophore, a fluorophor or an enzyme. The amount of tagged material bound immuno-chemically to the solid phase will depend upon the amount of untagged component in solution competing for the same binding sites. The more of the unknown present, the less will be the amount of tagged component bound.
In the second general method, termed the sandwich method or direct method, the solid phase containing an amount of immunochemically bound mobile component resulting from the first immunochemical reaction is subjected to the action of a reagent which can also bind immunochemically to the solid phase, but only at sites already occupied by the immunochemically bound mobile component. The reagent may be tagged, for example, as in the first method with a radioisotope, a fluorophor, a chromaphore or an enzyme. The amount of tagged reagent bound is a direct measure of the amount of mobile component bound, which, in turn, is a measure of the amount of mobile component initially present in the reaction mixture.
Where the tag is a radioisotope, the technique, whether competitive or noncompetitive, is termed a radioimmunoassay. When the tag is an enzyme, the assay is termed an enzyme-linked immunoassay. The amount of enzyme-tagged reactant is measured by any convenient method for measuring the activity of the enzyme used in the tag.
Other kinds of solid phase reactions of the ype generally described hereinabove are presented by way of example. The immunoradiometric assay for quantitative determination of an antigen is conducted by first reacting a known excess of labeled antibody with he unknown amount of antigen in a homogeneous phase reaction. Subsequently, immobilized antigen in excess amount is added in order to bind the unreacted soluble labeled antibody. The amount of unknown antigen is determined by measuring the difference between the total labeled antibody and the amount bound to the solid phase. The method gives direct quantitative results only with an univalent antigen, i.e., antigen which can only bind one molecule of antibody.
In such solid phase technology, the reagent or reagents used in the procedure are usually immobilized by being coated or bonded, either covalently or by adsorption to the solid phase material, which is then immersed in the sample to be tested. The manner of coupling such reagents to the solid phase material is known. See, for example, the disclosures in U.S. Pat. Nos. 3,652,761, 3,879,262 and 3,986,217.
A solid hase immunological assay system is disclosed in Miles et al., "Properties of Two-Site Immunoradiometric (Labelled Antibody) Assay Systems", IAEA, 149 (1974) in which solid-phase antibodies were bound to a tube wall by an immunoglobulin "arm". Polystyrene tubes were coated with non-immune guinea pig immunoglobulin (GP.IgG) or rabbit-anti (GP.IgG) (R-anti(GP.IgG)), and immunoglobulin "spacer arms" of various lengths were built up by alternative reactions with GP.IgG and R-anti (GP.IgG) leaving a final coat of the latter. Antibodies specific to glial fibrillary acidic protein (GP-anti(GFAP)) and to ferritin (GP-anti(ferritin)) were then immunologically bound to the solid phase. This placed specific solid-phase antibody at various distances from the matrix. Increasing "arm" length was shown to improve the precision of the dose-response variable.
U.S. Pat. No. 4,081,244, issued Mar. 28, 1978 to Polito et al. discloses a method for the preparation of an immunochemical composite comprising an antibody bound through a diamino spacer molecule to a finely divided polysaccharide matrix using a bifunctional coupling agent. The antibody coupled to the spacer may be either a primary or a secondary antibody, although the latter is preferred.
In U.S. Pat. No. 4,092,408 issued to Litt on May 30, 1978, a solid-phase radioimmunoassay method is disclosed in which anti-antibody is adsorbed on a solid surface and antibody is then bound to the anti-antibody. This immobilized antibody is then employed in a radioimmunoassay of antigen.
Examples of commonly used solid phase materials include, but are not limited to, glass or polymeric tubes which are coated with the reagent or reagents on their internal surfaces; coated polymeric inserts; coated polymeric sticks as disclosed in copending application Ser. No. 905,552 of Piasio et al., filed May 15, 1978 now U.S. Pat. No. 4,225,475; micro and macro beads formed of polymers and of glass; porous matrices; coated membranes; and tablets.
Immunochemical assays are highly useful in clinical research and diagnosis. They are highly specific, owing to the highly selective nature of antigen-antibody reactions. The antigen-antibody binding is very tight so that once the binding reaction has had an opportunity to occur, the limit of detectability is determined by the measurability with which the tag can be detected. Immunochemical assays are exceedingly versatile, owing to the fact that they can be used to measure specific substances selectively against a background of chemically similar substances. Because of these desirable attributes, there has been considerable interest in improving the ease of manipulation, sensitivity, accuracy, speed and applicability of immunochemical assays. The development of solid phase immunoassays has been one of the major advances in the field.
Among the advantages of solid phase systems is that the reaction product or products can be separated from the reaction solution with relative ease, i.e., by physically removing the solid phase material. This is in contrast with a non-solid phase or a homogeneous reaction, which typically results in a homogeneous solution which requires more complex separation techniques.
The introduction of solid phase technology has permitted the performance of novel procedures that were heretofore extremely difficult using free solution technology. An example of this is the sandwich assay technique described hereinabove. While a sandwich assay is theoretically possible in a homogeneous solution, it is not desirable for practical reasons. The most important aspect which makes such assays impractical is the separation of the first antigen-antibody complex from a homogeneous phase solution requires the use of sophisticated physical-chemical techniques, especially if the antigen is relatively small compared to the antibody and molecular weight differences between free antibody and complexed antibody are slight. In contrast, the separation procedure in a solid phase system is a matter of the utmost simplicity.
The earliest solid phase system devised were test tubes coated on the inside surface. Commercial examples of coated tube technology include the Immunotube.sup.tm system marketed by Smith Kline Instruments of Sunnyvale, California, and the Rianen.TM. system of New England Nuclear, North Billerica, Massachusetts, and the tubes described in U.S. Pat. No. 3,867,517 issued Feb. 18, 1975 to Ling. Although coated tube systems have proven useful for immunoassay purposes, they fail to exploit the full range of potential advantages offered by solid phase systems. A principle disadvantage is that the surface-to-volume ratio is relatively low and reaction kinetics may be further hindered by the fact that the reactive surfaces are located at the boundary of the solution volume, which may be relatively remote from the main body of the solution. Therefore, the average distance between mobile reactants and reactive surfaces is large.
The method of conducting in vitro diagnostic tests utilizing coated tubes is in widespread use. The disadvantages and defects of coated tubes are well known in the art. Many attempts have been made to improve on the performance of coated tubes with varying degrees of success. A description of several of these attempts will follow below. Some improvements have been made on the performance of coated tubes, but the search for better solid phase systems still continues. One improvement is he coated polymeric sticks as disclosed in copending application Ser. No. 905,552 now U.S. Pat. No. 4,225,475. Applicants have improved on the performance of these sticks by conducting the test in a receptacle which has the same coating as on the sticks. For example, a coated tube requires 70 minutes to reach 50% of equilibrium for digoxin assay. A coated stick, finned as described in application Ser. No. 905,552, on the other hand, requires 25 minutes. By utilizing such a coated finned stick and conducting the assay in a coated tube, 50% of equilibrium is reached in only 10 minutes--an improvement over both of the other two systems.
Attempts to improve on the performance of coated tubes have led to a variety of systems designed to increase the surface to volume ratio of the solid phase system. These methods have included providing highly convoluted surfaces, of finely divided material.
The SPAC.sup..TM. system of Mallinckrodt Chemical Company is basically a coated tube system which exemplifies the strategy of providing a convoluted surface to increase surface area in the coated tube format. Additionally, the tubes are provided with a detachable lower section which may be batch coated to achieve greater uniformity from tube to tube.
A consequence of the batch immobilization on coated tube bottoms is that the outside as well as the insides of the tube become coated. This makes it difficult for the laboratory technician to wwork with the tubes without coming into contact with whatever materials are coated on their surface and valuable immunological reactants are wasted. The convoluted surface area is said to increase by hree to four times the amount of reactive surface available. However, the reactive surface remains at the periphery of the solution, which may be suboptimal geometry from the standpoint of the average diffusion distance from the solution to the reactive surface. Due to the complexity of the surface, difficulties in washing the surface free of contaminating substances may be encountered. As with coated tube systems in general, the SPAC.sup.tm system is likely to be sensitive to convection currents which can result in large errors as previously described. Convection may be reduced by carrying out the reaction in a constant temperature bath. However, this procedure presents additional equipment requirements for the clinical laboratory. For measurement of hapten antigens, the system is additionally suboptimal if the reaction is carried out at 37.degree. C. according to the manufacturer's recommendation. It has been shown that increasing the temperature of a certain antibody-hapten reaction tends to enhance the rate of dissociation of the antibody-hapten complex relative to the rate of its formation. See Smith, T. W., and Skubitz, K. M., Biochemistry 14, 1496 (1975) and Keave, P. M., Walker, W. H. C., Gauldie, J. and Abraham, G. E., Clinical Chemistry 22, 70 (1976).
Various types of solid phase matrices designed to be inserted into the reaction fluid have been disclosed. A convoluted or sponge-like matrix designed to be inserted into the test solution is exemplified by U.S. Pat. No. 3,951,748, issued Apr. 20, 1976 to Devlin. This material offers relatively large surface areas but may be difficult to wash or drain thoroughly at the conclusion of the reaction. In addition, such systems may be limited in practice to the use of reactants and reagents which are readily eluted from the sponge matrix. More significantly, the sponge matrices tend to react extensively with only a portion of the reaction fluid, i.e., that portion which actually penetrates the pores of the matrix. Another solid phase matrix useful for assaying biologically active materials is disclosed in U.S. Pat. No. 4,066,512, issued Jan. 3, 1978 to Lai et al. This matrix comprises a microporous membrane, an inert proteinaceous material coated thereon, and a biologically active material immobilized onto this coating. This matrix can then be used for determining an unknown in a fluid sample.
A second type of insert, employing the strategy of forcing the reaction fluid to spread in a thin layer over the coated matrix surface, is disclosed in U.S. Pat. No. 3,826,619, issued July 30, 1974 to Bratu, et al., and U.S. Pat. No. 3,464,798, issued Sept. 2, 1969 to Kilthau. Both cases disclose a combination of a receptacle and closely-fitting insert matrix, so shaped as to sqeeze the reaction fluid into a thin layer between the container walls and the matrix surface. The insert matrix must fit the container with a close tolerance, and the volume of reaction fluid must be carefully controlled, since variations could adversely affect the reproducibility of the assay. The apparatus of Bratu is designed for use in a direct immunochemical test that is qualitative only. Because the reaction solution is forced into a thin film by the insert, the reaction volume must necessarily be small and Bratu in fact discloses that the type of assay contemplated is designed for small volumes of undiluted serum. One of the pitfalls of this type of assay is that errors in the rates of antigen-antibody reactions may be caused by variations in the pH of undiluted serum, which may vary between pH 6 and pH 9 in clinical samples. The pH may be controlled by the addition of a buffer, but buffer salt concentrations greater than 0.1 M tend to dissociate antigen-antibody complexes. Therefore, an excess volume of low ionic strength buffer must be used to control pH accurately, and this may expand reaction volume to an unacceptable amount. Error due to the pH may be tolerated in a qualitative assay such as disclosed by Bratu et al., especially in samples relatively rich in concentration of unknown, but not in the quantitative assays for which the present invention is designed. Where diluting by buffer is required, a low concentration of unknown may be diluted below the level of detection, leading to false negative results with the Bratu or Kilthau device. A false negative result is one in which no unknown is detected when some should have been detected. One embodiment of the Bratu insert is an insert having four fins. Its use is disclosed for qualitative analysis where larger quantities of serum are available, but there is no suggestion of any different mode of operation from the thin film mode utilized with the rounded or conical version. The devices disclosed in U.S. Pat. No. 3,826,619 have not, so far is known, been commercially exploited.
An example of an insert which utilizes the "intimate contact" principle of Bratu and Kilthau, but is apparently used quantitatively, is disclosed in U.S. Pat. No. 4,135,884 issued Jan. 23, 1979 to Shen and represented by the "Gamma Stick.TM. [.sup.125 I] T.sub.3 Uptake Kit" of Alpha Gamma Labs, Inc., Sierra Madre, California. This insert has four flutes which are coated with an antigen or antibody and inserted into a test tube containing the unknown sample, in intimate contact with the sample.
A third type of solid phase insert matrix is represented by the StiQ.TM. assay of International Diagnostic Technology Corporation, Santra Clara, California, designed to exploit a solid phase assay disclosed in U.S. Pat. No. 4,020,151, issued Apr. 26, 1977 to Bolz, et al. In this system, a disc shaped, uncoated insert maxtrix of material capable of adsorbing proteins from serum is provided. In this system, the limitations are not only due to surface-to-volume ratio or geometric considerations but are mainly due to problems associated with the initial adsorption step, such as the presence of interfering substances and the difficulty of obtaining measurable adsorption components present in low concentration.
Another example of an attempt to improve surface-to-volume ratio by reducing reaction volume is disclosed by Friedel, R. and Dwenger, A., Clin Chem. 21 967 (1975). In this system, capillary tubes are coated on the inside with a specific adsorbant and the reaction mixture is introduced into the lumen of the capillary tube.
A further example of such a device is disclosed in U.S. Pat. No. 4,111,754, issued Sept. 5, 1978 to Park which relates to a solid phase matrix having a cylindrical supporting surface with inwardly directing protuberations. The spacing between the protuberations is such that a liquid sample will be retained within the matrix by capillary action so that the sample can only be removed from the matrix by addition of another fluid on top of the matrix to build up a hydrostatic pressure head sufficient to overcome the capillary attraction. A further example of the continuing prior art trend toward maximizing surface to volume ratio and capillary devices is shown by U.S. Pat. No. 4,116,638 issued Sept. 26, 1978 to Kenoff. The Kenoff device consists of a bundle of capillary tubes contained in a holder designed to be inserted into a sample contained in a test tube.
One system which affords a high surface area for overall volume is the coated micro glass bead system as, for example, the Immo Phase.TM. system of Corning Glass Works. This system exemplifies the use of finely divided particles. It provides a high coated surface area with a correspondingly high reaction rate. Due to settling of the particles during the reaction, optimization of test systems of this kind require that the test tubes in which they are placed during reaction be capped and mixed vertically during reaction to insure that all surfaces come in contact with the reactants. Further, the use of particles necessitates multiple centrifugations and washings to completely separate the immobilized product from the solution.
Another example of a system which affords high surface area for over-all volume is the coated macro bead as disclosed in U.S. Pat. No. 3,932,141, issued Jan. 13, 1976, to Beall et al. and represented by the AUSRIA.sup.tm II-125 and AUSAB.sup.tm assays of Abbott Laboratories, North Chicago, Ill. This bead is designed so that a minimal amount of sample is required. It appears that the sample forms a thin film around the bead and as such would have the same deficiencies as described above for the Bratu device. This bead appears to be only useful for qualitative analysis and not for quantitative analysis.
Other examples of solid phase matrices, which alleviate many of the deficiencies of the prior art, are disclosed in copending patent application Ser. No. 805,431, filed June 10, 1977 of Piasio et al. now U.S. Pat. No. 4,197,287 and copending patent application Ser. No. 905,552 of Piasio et al., filed May 15, 1978 now U.S. Pat. No. 4,225,475. Application Ser. No. 805,431 discloses a water-insoluble solid phase matrix for insertion into a reaction vessel which comprises an elongated annular support surface and a plurality of water-insoluble fins projecting from the support surface. An antigen or an antibody capable of reacting with a mobile component in a liquid sample to be assayed is immobilized on the interior of the annular surface and on each of the fin surfaces. Application Ser. No. 905,552 discloses another water-insoluble solid phase matrix for insertion into a reaction vessel which comprises a handle having a plurality of essentially smooth curved or planar surfaces attached thereto which extend throughout the liquid sample being assayed. An antigen or an antibody is immobilized on the curved or planar surfaces which reacts with a mobile component in the liquid sample.
Prior attempts to improve on coated tubes as a solid state reaction matrix have frequently resulted in some improvement in reaction rate or the time necessary to carry out a measurable reaction. Such improvement has often been accomplished by concomitant increase in manipulative difficulty, or loss of flexibility.